Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.     

Identification of a new P450 expressed in human lung: complete cDNA sequence, cDNA-directed expression, and chromosome mapping

A cDNA coding for a P450 expressed in human lung was isolated from a lambda gt11 library constructed from human lung mRNA using a cDNA probe to rat P450 IVA1. The cDNA-deduced amino acid sequence of this P450, designated IVB1, consisted of 511 amino acids and had a calculated molecular weight of 59,558. The IVB1 amino acid sequence bore 51%, 53%, and 52% similarities to rat IVA1, IVA2, and rabbit P450p-2, respectively. Comparison of the primary amino acid sequence of human IVB1 with rat IVA and rabbit p-2 P450 sequences revealed a region of absolute sequence identity of 17 amino acids between residues 304 and 320. However, the functional significance of this conserved sequence is unknown. Human IVB1 also appears to be related to P450 isozyme 5 that has been extensively characterized in rabbits. The IVB1 cDNA was inserted into a vaccinia virus expression vector and the enzyme expressed in human cell lines. The expressed enzyme had an absorption spectrum with a lambda max at 450 nm when reduced and complexed with carbon monoxide, typical of other cytochrome P450s. Unlike rabbit P450 isozyme 5, however, human IVB1 was unable to activate the promutagen 2-aminofluorene. Human lung microsomal P450s were also unable to metabolize this compound despite the presence of IVB1 mRNA in three out of four human lungs analyzed. In contrast to its expression in lung, IVB1 mRNA was undetectable in livers from 14 individuals, including those from which the lungs were derived. IVB1-related mRNA was also expressed in rat lung and was undetectable in untreated rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a novel P450 expressed in rat lung: cDNA cloning and sequence, chromosome mapping, and induction by 3-methylcholanthrene

A novel P450 cDNA was isolated from a rat lung lambda gt11 library by hybridization with the rat P450 IIB1 cDNA probe. The cDNA-deduced amino acid sequence of this clone was 71% and 73% similar to rat IIA1 and IIA2 P450s; it was, therefore, designated IIA3 as the third member of the rat IIA subfamily. IIA3 demonstrates only 55% amino acid similarity with IIB1. Interestingly, this P450 also shared 85% and 94% amino acid similarities with human IIA3 and a mouse testosterone 15 alpha-hydroxylase P450, respectively, indicating that these P450s are orthologous counterparts to rat IIA3. Chromosome mapping, using mouse-hamster somatic cell hybrids, revealed that the IIA3 gene is localized on mouse chromosome 7. The IIA3 mRNA was detected in rat lung, and its level was induced 3-fold by treatment of rats with 3-methylcholanthrene. No IIA3 mRNA was seen in the liver, kidney, or intestine, even after long exposure of Northern blot filters to X-ray film. In contrast, the orthologous mouse and human IIA3 genes are expressed in liver.     

cDNA cloning and regulation of a novel rat cytochrome P450 of the 2C gene subfamily (P450IIC24)

A novel member of the cytochrome P450 2C gene subfamily was identified by screening rat prostate cDNA libraries. Two independent clones were isolated. Clone pros1 was 1031 bp long and contained a bizarre replacement in place of putative exon 1. Clone pros2 was 1755 bp long, contained a complete 3' end, and also had bizarre sequences in place of exon 1, which in this case were compatible with an unspliced intron. Northern analysis revealed mRNA expression in the liver and the kidney. Interestingly, although livers of mature rats of both sexes have comparable amounts of P4502C24 mRNA, a dramatic sex difference is seen in the kidney where only males express detectable levels of this mRNA.     

Molecular cloning of chicken metallothionein. Deduction of the complete amino acid sequence and analysis of expression using cloned cDNA.

A cDNA library was constructed using RNA isolated from the livers of chickens which had been treated with zinc. This library was screened with a RNA probe complementary to mouse metallothionein-I (MT), and eight chicken MT cDNA clones were obtained. All of the cDNA clones contained nucleotide sequences homologous to regions of the longest (376 bp) cDNA clone. The latter contained an open reading frame of 189 bp, and the deduced amino acid sequence indicates a protein of 63 amino acids of which 20 are cysteine residues. Amino acid composition and partial amino acid sequence analyses of purified chicken MT protein agreed with the amino acid composition and sequence deduced from the cloned cDNA. Amino acid sequence comparisons establish that chicken MT shares extensive homology with mammalian MTs, but is more closely related to the MT-II than to the MT-I isoforms from various mammals. The nucleotide sequence of the coding region of chicken MT shares approximately 70% homology with the consensus sequence for the mammalian MTs. Southern blot analysis of chicken DNA indicates that the chicken MT gene is not a part of a large family of related sequences, but rather is likely to be a unique gene sequence. In the chicken liver, levels of chicken MT mRNA were rapidly induced by metals (Cd2+, Zn2+, Cu2+), glucocorticoids and lipopolysaccharide. MT mRNA was present in low levels in embryonic liver and increased to high levels during the first week after hatching before decreasing again to the basal levels found in adult liver. The results of this study establish that MT is highly conserved between birds and mammals and is regulated in the chicken by agents which also regulate expression of mammalian MT genes. However, in contrast to the mammals, the results suggest the existence of a single isoform of MT in the chicken.     

Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2

Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.     

Nucleotide and deduced amino acid sequence of the E1 alpha subunit of human liver branched-chain alpha-ketoacid dehydrogenase

A 1552-bp cDNA for the E1 alpha subunit of branched-chain alpha-ketoacid dehydrogenase (BCKDH) was isolated from a human liver cDNA library. The cDNA contained a 1134-bp open reading frame that encoded 378 amino acid (aa) residues of the enzyme and 418 bp of 3'-untranslated sequence. The deduced amino acid sequence of the human protein shows 96% identity with that of the rat enzyme subunit. Those 117-aa residues surrounding the phosphorylation sites are completely conserved between man and rat. BCKDH E1 alpha showed considerable amino acid sequence similarity with pyruvate dehydrogenase E1 alpha, particularly in the region of the two principal phosphorylation sites of these proteins. Northern blots of human liver and skin fibroblasts demonstrated a single 1.8-kb mRNA band, with a higher level of E1 alpha mRNA in liver than in normal fibroblasts. Fibroblasts from a patient with thiamine-responsive maple syrup urine disease (MSUD) contained an mRNA of the same size and abundance as that of normal fibroblasts. Genomic DNA from normal and MSUD fibroblasts gave the same restriction maps on Southern blots, and the gene was approximately 10-kb in size.     

Isolation and characterization of full-length mouse cDNA and genomic clones of 3-methylcholanthrene-inducible cytochrome P1-450 and P3-450

Polysome immuno-adsorption, with immunoglobulin G directed against two 3-methylcholanthrene-induced mouse liver cytochrome P-450 proteins, was used to enrich mRNA from 3-methylcholanthrene-treated C57BL/6N mouse liver. cDNA transcribed from the P-450-enriched mRNA was then cloned into the Okayama-Berg vector. Two cDNA classes were detected upon differential screening of the clone bank with [32P]cDNA derived from 3-methylcholanthrene-induced immuno-enriched versus control mRNA. Several representatives of these two classes were judged to be near full length by comparison with their corresponding mRNA mobilities on denaturing agarose gels. A continuous reading-frame near the 5' end of one cDNA class (P1-450) corresponds to a protein having 15 of 17 residues the same as the published N-terminal sequence of rat P-450c. A continuous reading frame near the 5' end of the other class (P3-450) corresponds exactly to the first 25 amino acids of the published N-terminal sequence of rat P-450d. The P1-450 cDNA is at least 700 bp longer than the P3-450 cDNA. Heteroduplex analysis and Southern blot hybridization demonstrate that these mRNAs share approx. 1100 bp of sequence homology. Genomic P1-450 and P3-450 clones were isolated from a gene library constructed from C57BL/6N mouse liver DNA. By heteroduplex analysis with the corresponding cDNA, the P1-450 gene spans about 6 kb and the P3-450 gene about 7 kb. The intron-exon patterns are very similar, with the second and seventh exons being much larger than the other five. The 3' terminal exon of P1-450 is about 500 bp longer than that of P3-450. These data suggest that both P1-450 and P3-450 have diverged from a common ancestral gene.     

Characterization of a cDNA for rat P-450g, a highly polymorphic, male-specific cytochrome in the P-450IIC subfamily

Cytochrome P-450g (IIC13) is a highly polymorphic, male-specific rat liver isozyme which is a member of the P-450IIC subfamily. A cDNA, c5126 (1737 bp), for P-450g was isolated from a lambda gt11 library synthesized from (+g) male rat liver mRNA. Sequence analysis of the clone, c5126, revealed an open reading frame of 1473 nucleotides, which encodes for a 490 amino acid polypeptide possessing the 30 NH2-terminal residues reported for cytochrome P-450 (M-3) (P-450g) [Matsumoto et al. (1986) J. Biochem. 100, 1359-1371]. A high degree of sequence similarity (greater than 70%) exists between c5126 and the published sequences of cDNAs for members of the IIC subfamily, while its sequence similarity to other subfamilies (IA, IIB, and IIIA) was much lower (less than 55%). RNA blot analysis utilizing an oligonucleotide probe specific for P-450g revealed that P-450g mRNA was expressed in livers of male but not female Sprague-Dawley (CD) and ACI rats, indicating that the sex difference was regulated pretranslationally. Furthermore, expression of P-450g mRNA was age dependent in livers of male ACI rats (a homozygous, phenotypically high P-450g strain). However, the mRNA for P-450g was expressed equally in livers of outbred male CD rats representing either the high (+g) or the low (-g) phenotype and of inbred ACI rats (+g) representing the high phenotype, indicating that the defect in (-g) rats does not reflect differences in expression of P-450g mRNA.     

cDNA cloning of cytochrome P-450 related to P-450p-2 from the cDNA library of human placenta. Gene structure and expression

We have isolated and analyzed cDNA (designated P-450HP cDNA) clones from a human placenta cDNA library, using the cDNA for rabbit pulmonary cytochrome P-450p-2, a prostaglandin omega-hydroxylase, as a hybridization probe. The cDNA obtained encoded a polypeptide comprising 511 amino acids with a calculated molecular mass of 58987 Da, and the amino acid sequence similarity with P-450p-2 and rat liver laurate omega-hydroxylase (P-450LA omega) was only about 50%. RNA blot analysis showed that the mRNA hybridizable with the human P-450HP cDNA was inducibly expressed 3-5-fold in rabbit small intestine and lung by gestation, but the expression remained constant in rabbit liver and kidney. This mode of expression was quite different from that of P-450p-2 and P-450LA omega. Interestingly, the mRNA hybridized with the cDNA of P-450HP was found to be expressed in all the human tumor tissues so far examined, in sharp contrast with the facts that almost all the other species of P-450s are known to disappear in the tumor tissues. Taken together, the deduced hemoprotein termed P-450HP dose not seem to be the human counterpart of rabbit P-450p-2 or rat P-450LA omega, and is presumably a new member of the P-450 family including P-450p-2 and P-450LA omega. Furthermore, the corresponding genomic DNA was also cloned and analyzed. The gene of P-450HP spanned 18.8 kb and was separated into 11 exons by 10 introns whose locations were completely different from those of P-450 genes so far determined.     

Induction of cytochrome P-450IA1, IA2, IIB1, IIB2 and IIE1 by broccoli in rat liver and colon

Ingestion of broccoli or other cruciferous vegetables inhibits the induction of cancer by chemicals and modifies some cytochrome P-450 enzyme activities. The effect of dietary broccoli on the levels of P450IA and IIB mRNA and proteins in rat liver and colon has been studied. Rats were fed a ten percent broccoli diet for 7 days. The expression of the cytochrome P-450 forms was altered to a different extent in the liver and colon. The level of total P450IA mRNA in the liver was increased by the broccoli together with the P450IA1 and IA2 proteins. Colonic P450IA1 mRNA and protein were induced by the broccoli diet, whereas only P450IA2 protein and not mRNA was detectable in colon, but the protein level was unaffected by the broccoli diet. Liver P450IIB and IIE1 proteins were increased by the broccoli diet, whereas the level of P450IIB mRNAs was not affected. In contrast, the P450IIB mRNA levels were reduced but the protein levels were increased in colon and we suggest that a feedback mechanism caused the decrease of the P450IIB mRNAs levels. Because the ratio between activation and deactivation may be an important risk determinant, we conclude that the protective effect of the broccoli diet on chemically induced tumors in rodents may be caused by the broccoli-induced changes in P450IA and IIB associated enzyme activities.     

Molecular cloning and sequence analysis of cDNA for the catalytic subunit 1 alpha of rat kidney type 1 protein phosphatase, and detection of the gene expression at high levels in hepatoma cells and regenerating livers as compared to rat livers.

A cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit 1 alpha has been isolated from a rat kidney lambda gt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase 1 alpha from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type 1 alpha protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2A alpha protein phosphatase. Thus, the protein sequence of type 1 alpha protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of AH13 were remarkably increased when compared to those of normal ivers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.     

Primary structure of rat liver alkaline phosphatase deduced from its cDNA.

Rat liver alkaline phosphatase (ALP) was markedly induced by treatment of rats by bile-duct ligation and colchicine injection. Taking this advantage for enrichment of ALP mRNA, we constructed a lambda gt11 liver cDNA library using polyadenylated RNA prepared from the treated rat liver, and isolated an ALP cDNA clone. The 2165 bp cDNA contained an open reading frame that encodes a 524-amino-acid-residue polypeptide with a predicted molecular mass of 57737 Da. The precursor protein contained a presumed signal peptide of 17 amino acid residues followed by 28 amino acid residues identical with the N-terminal sequence determined from the purified rat liver ALP. It was also confirmed that amino acid sequences of two CNBr-cleavage peptides obtained from liver ALP were contained within the cDNA-encoded protein. Five possible N-linked glycosylation sites were found in the molecule and a highly hydrophobic amino acid sequence at the C-terminus. The deduced polypeptide of rat liver ALP showed 88% homology to that of the human liver-type enzyme in osteosarcoma cells. RNA blot hybridization analysis identified a single species of ALP mRNA with 2.7 kb in both the control and the treated rat livers. An approx. 20-fold increase of the mRNA was detected in the treated liver at 12 h after the onset of stimulation, compared with that in the control liver.     

Cloning and characterization of two major 3-methylcholanthrene inducible hamster liver cytochrome P450s

We have studied the immunochemical properties of two major 3-methylcholanthrene inducible hamster liver cytochrome P450 isozymes, P450 MC1 and P450 MC4. Immunoblots using specific antibodies against P450 MC1 and P450 MC4 demonstrated that these two P450s were present in very low levels in control hamster livers and were greatly induced by 3-methylcholanthrene treatment. P450 MC1 was immunochemically different from P450 MC4, rat P450c and P450d, and rabbit LM4. The immunorelated polypeptide to P450 MC1 was not present in the control or the 3-methylcholanthrene-treated rat liver microsomes, whereas it was present in two human liver microsomal preparations. On the other hand, P450 MC4 was immunochemically related to rat P450d and rabbit LM4. The immunorelated polypeptide to P450 MC4 was present in the human and 3-methylcholanthrene-treated rat liver microsomes. We also isolated full-length cDNA clones encoding P450 MC1 and P450 MC4 mRNAs from a 3-methylcholanthrene-induced hamster liver cDNA library. The full-length cDNA clones of P450 MC1 and P450 MC4 contained 1771 and 1868 base pairs, which encoded polypeptides of 494 and 513 amino acids, respectively. RNA blot analysis revealed that the mRNAs for P450 MC1 and P450 MC4 were 2100 and 2600 bases in length, respectively. 3-Methylcholanthrene pretreatment increased the P450 MC1 mRNA level by 16-fold and the P450 MC4 mRNA level by 11-fold in the hamster livers. A comparison of the deduced amino acid sequences with other cytochrome P450s revealed that P450 MC1 was most similar to the mouse P450(15) alpha with 75% sequence identity, whereas P450 MC4 shared 87% identity with the rat P450d or mouse P3(450). These results indicated that P450 MC1 was a unique member (CYP2A8) in the P450IIA subfamily, whereas P450 MC4 was the hamster P450IA2.     

Rat testosterone 7 alpha-hydroxylase. Isolation, sequence, and expression of cDNA and its developmental regulation and induction by 3-methylcholanthrene

A P-450, designated P-450a, with high testosterone 7 alpha-hydroxylase activity was purified from rat liver microsomes. Specific polyclonal antibody against this P-450 was used to screen a lambda gt11 expression cDNA library and a 1687-base pair cDNA was isolated and sequenced. The deduced protein had 492 amino acids, a calculated Mr of 56,016, and it shared 51 and 45% amino acid similarities to P-450e and P-450f, respectively. Regions of similarity were distributed in distinct areas of high and low similarity along the P-450a primary sequence. P-450a cDNA was introduced into yeast cells using the expression vector pAAH5, and the resultant yeast microsomes contained both a protein of identical electrophoretic mobility to that of rat P-450a and testosterone 7 alpha-hydroxylase activity. These results confirm enzyme reconstitution data and antibody inhibition data that P-450a possesses testosterone 7 alpha-hydroxylase activity. The antibody and cDNA probes were used to examine the mechanism of regulation of P-450a by inducers and during development. P-450a and its mRNA were present at low level in newborn rats and increased to maximal level at 1 week of age in both males and females. At age 12 weeks, however, the P-450a level decreased in males but remained elevated in females. Concomitant with the decrease in P-450a in adult males was an increase in level of another immunologically related P-450. In adult male rats, P-450a was induced almost 5-fold by administration of 3-methylcholanthrene and this induction was the result of an increase in its mRNA. These results establish testosterone 7 alpha-hydroxylase as a member of the P-450e gene family that is developmentally regulated, sex-dependent, and markedly inducible by 3-methylcholanthrene.